PROJECT SUMMARY/ABSTRACT The tumor microenvironment (TME) is the chemical and biological background that affects tumor malignancy, proliferation and metastatic activity because of chaotic angiogenesis tumors growing to become less oxygenated (low pO2) and more acidic (low pH). While pO2 and pH are known to be important factors for tumor growth and treatment, interstitial Pi has been recently identified as a new signaling molecule of importance in tumorigenesis. Newly synthesized stable paramagnetic molecules, spin probes, permit simultaneous in vivo monitoring of interstitial pO2, extracellular pH (pHe) and concentration of interstitial inorganic phosphate (Pi) using Electron Paramagnetic Resonance (EPR). Because tumors are highly heterogeneous, spectral-spatial EPR imaging is required to spatially resolve the parameters of interest. Spatial resolution is defined by EPR sensitivity. The amount of the spin probe molecules within a voxel decreases as spatial resolution is cubed, and may become undetectable if too small. Sensitivity also directly translates into functional resolution, the accuracy with which pH, pO2, and Pi can be measured. The proposal by the PI and his colleagues for a Rapid Scan (RS) EPR technique has been explored at 250 MHz frequency and showed improvement in signal-to-noise ratio up to two orders of magnitude compared to the standard field-modulated continuous wave (CW) method. An additional order of magnitude signal enhancement can be achieved for trityl-based multifunctional probes by increasing EPR frequency to 700 -800 MHz (optimal for small animal RS EPR studies) and scan frequency to 50-100 kHz (the current mathematical algorithm limits the upper scan frequency to about 10 kHz). RS EPR sensitivity enhancement will require both hardware (SA1) and software (SA2) developments. In addition, novel 4D spectral- spatial algorithm will be further developed to enable multi-functional multi-line EPR imaging, since the standard filtered backprojection (FBP) reconstruction fails to work with multi-functional spin probes that have a comparatively broad multi-line EPR spectra. A newly developed multi-functional EPR imager will first be tested in vitro on standard samples with known geometry, pO2, pH, and Pi patterns, followed by in vivo proof-of-concept imaging. A colony of PyMT transgenic mice will be used that spontaneously-develop breast cancer to perform the rapid scan multifunctional imaging and construct spatially-resolved pO2, pH, and Pi profiles of TME and normal mammary gland. We anticipate to achieve physiologically significant voxel-specific functional sensitivities of about 1-2 mmHg of pO2, 0.05 pH units and 0.1 mM of inorganic phosphate. Estimated spatial resolution will be smaller than 200 ?m. In summary, the success of this project may have a significant impact on the future of in vivo functional imaging to study TME and beyond.